RESUMEN
STUDY DESIGN: This study was designed to investigate the effects of oxidant and antioxidant treatment, as well as regular exercise, on neurotrophin levels in the spinal cord of rats. OBJECTIVES: Reactive oxygen species (ROS) play a role in neurodegenerative diseases, but ROS at moderate levels could stimulate biochemical processes through redox-sensitive transcription. METHODS: Exercised or sedentary animals were injected subcutaneously with hydrogen peroxide (H(2)O(2)), N-tert butyl-alpha-phenyl nitrone (PBN) or saline for the last 2 weeks of a 10-week experimental period to challenge redox balance. Free radical (FR) concentration was evaluated in the spinal cord by electron spin resonance, protein carbonyls, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) levels and the mRNA expression of BDNF receptor and tyrosine kinase receptor B (TrKB). SETTING: Research Institute of Sport Science, Semmelweis University, Budapest, Hungary. RESULTS: Exercise or PBN decreased the concentration of FR, whereas the carbonyl content did not change. BDNF was significantly decreased in exercised sham and sedentary PBN-treated groups, and its content correlated with the level of FR. GDNF was significantly increased in sedentary H(2)O(2)-treated groups. No differences were observed in TrkB mRNA expression among groups. CONCLUSIONS: Results suggest that regular exercise alone and PBN in sedentary animals can successfully decrease FR levels in the spinal cord. Redox alteration seems to affect the levels of GDNF and BDNF, which might have clinical consequences, as neurotrophins play an important role in cellular resistance and regeneration.
Asunto(s)
Antioxidantes/farmacología , Factores de Crecimiento Nervioso/metabolismo , Oxidantes/farmacología , Condicionamiento Físico Animal/métodos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Análisis de Varianza , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Óxidos N-Cíclicos/farmacología , Radicales Libres/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Peróxido de Hidrógeno/farmacología , Masculino , Factores de Crecimiento Nervioso/clasificación , Factores de Crecimiento Nervioso/genética , Carbonilación Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
A possible molecular mechanism for the constitutive activity of mutants of the angiotensin type 1 receptor (AT1) at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT(1) receptor reveals a novel nonsimplistic view on the general problem of constitutive activity, and clearly demonstrates the inherent complexity of the process of G protein-coupled receptor (GPCR) activation.
Asunto(s)
Sustitución de Aminoácidos , Mutación , Receptor de Angiotensina Tipo 1/química , Receptores Acoplados a Proteínas G/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Membranas Intracelulares/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/métodos , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína/genética , Ratas , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/genética , TransfecciónRESUMEN
To study the role of extracellular-signal-regulated kinase (ERK) cascade and the small GTP-ase proteins in the activation of the c-fos promoter by angiotensin II (AII), transient transfection experiments were performed in CHO cells stably expressing the rat AT(1A) receptor. In this system AII activated ERK in 1 min and also increased the transcriptional activity of the c-fos promoter-luciferase reporter gene construct. The activation of the promoter proved to be dependent on the Ras-Raf-ERK cascade as cotransfection of expression vectors known to specifically inhibit this cascade blocked the effect of AII. Dominant-negative p21Rac1 mutant partially blocked the activation of the c-fos promoter by AII. However, activation of the c-fos promoter was independent of protein kinase C (PKC) as bisindolylmaleimide I, a specific PKC inhibitor did not block the effect of AII. These results suggest that AII activates the transcription of the c-fos through the Ras-Raf-ERK cascade. Furthermore, p21Rac1 is involved in the modulation of the c-fos promoter by AII.
Asunto(s)
Angiotensina II/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Transcripción Genética/fisiología , Proteína de Unión al GTP rac1/fisiología , Animales , Células CHO , Cricetinae , Fosfatidilinositol 3-Quinasas/fisiología , Regiones Promotoras Genéticas/fisiología , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-raf/fisiología , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/fisiología , Proteínas ras/fisiologíaRESUMEN
The agonist-induced phosphorylation sites of the rat AT1a angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta342) but was abolished by truncation at Ser325 (Delta325). Truncation at Ser335 (Delta335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by approximately 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta325 < Delta335 < ST-AA < Delta342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta325 > Delta335 > ST-AA = Delta342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.
Asunto(s)
Citoplasma/metabolismo , Receptores de Angiotensina/agonistas , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Células COS , Citoplasma/química , Fosfatos de Inositol/biosíntesis , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Serina/análisis , Treonina/análisis , TransfecciónRESUMEN
For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1 angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1, Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.
Asunto(s)
Asparagina/metabolismo , Receptores de Angiotensina/química , Alanina/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Células COS , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Fosfatos de Inositol/metabolismo , Mutagénesis , Estructura Secundaria de Proteína , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMEN
A series of deletion mutants was created to analyze the function of the membrane-proximal region of the cytoplasmic tail of the rat type 1a (AT1a) angiotensin receptor. In transiently transfected COS-7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar1,Ile8]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II-induced inositol phosphate responses was observed. Alanine substitutions in the region 307-311 identified the highly conserved phenylalanine309 and adjacent lysine residues as significant determinants of AT1a receptor expression.